Programmed cell death is a mechanism by which cells kill themselves in a regulated way. Studies in the nematode worm C. elegans (all you 219 students should have fond memories of this little worm!) led to the discovery of the first genes regulating programmed cell death. Scientists wondered where 131 cells present in the last larval stage went as they were not present in the adult. These investigations led to the 2002 Nobel Prize in Medicine—the first of 3 for C. elegans!
Apoptosis is also required in higher eukaryotes for proper development. Without programmed cell death we would all have webbed fingers and toes! Some people with a defect in this pathway still do! Apoptosis also regulates some aspects of metamorphosis. We are all familiar with the tadpole "losing" its tail to change into a frog.
Apoptosis occurs in our bodies every day. The reason our tissues and organs do not grow out of control is the delicate balance between apoptosis and cell division. When one or both of these processes are not under tight control cancer can develop.
Apoptosis is the most common form of programmed cell death (PCD), a process by which a cell dies by undergoing a series of well studied morphological changes. The cells characteristically shrink, nuclear material condenses and fragments, the cytoskeleton collapses, and internal membranes disassemble. The cells also change shape, "blebbing" off membrane-bound parts. These cells are rapidly recognized by macrophages, engulfed and eliminated. As a result, this type of cell death does not elicit an inflammatory response. Another form of cell death called necrosis occurs as a result of cell trauma whereby cells burst and spill their contents out onto neighboring cells. This results in an inflammatory response that may be damaging to neighboring cells.
The system we'll be using to study the process of apoptosis in this three-part lab series is the HL-60 cell line, which is a line of partially differentiated cells in the granulocyte blood cell lineage that was derived from a patient with acute promyelocytic leukemia. In contrast to the 3T3 fibroblast cells you used last week, which formed an attached monolayer on the bottom of a tissue culture flask, HL-60 are suspension cells that grow unattached to any surface. (This should make sense to you based on their blood cell lineage.) HL-60 cells are particularly sensitive to apoptotic stimuli, so they are commonly used to study the events of apoptosis. The particular apoptotic stimulus we'll use is a drug called etoposide (also known as VP-16), which inhibits the enzyme topoisomerase II, an essential player in DNA replication and repair. Cells undergo apoptosis as a response to widespread DNA damage. You'll be using cells that have been treated with etoposide for different periods of time. In today's lab, you'll observe the time-dependence of one feature of apoptosis, DNA fragmentation. Although other agencies consider HL-60 cells to be a Biosafety Level 1 (BL1) cell line, Wellesley College has designated them to be BL2. Therefore, we cannot allow you to work with living cells in a course lab. You will instead be given etoposide-treated, fixed cells from which you will extract the DNA and analyze its size distribution by agarose gel electrophoresis.
Genomic DNA Isolation Protocol
Cells were treated with either 500 uM etoposide (VP-16) dissolved in dimethyl sulfoxide (DMSO) or DMSO alone as a control over a time course of 8 hours. Samples were taken at 2-hour intervals (0, 2, 4, 6 and 8). At each interval 1x106 cells were pelleted in a microcentrifuge.
The following three steps have been done for you.
- Cells were washed twice with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.2).
- Washed cells were resuspended in DNA extraction buffer (50 mM Tris, pH 8.0, 20 mM Na2EDTA, 10 mM NaCl, 1% [w/v] SDS, and 20 μg/ml RNase A) and incubated at 37°C for 1 hour to lyse the cells and digest cellular RNA.
- Proteinase K was added to a final concentration of 100 ug/ml and incubated at 65°C for 1 hour to digest cellular proteins.
To do in lab in the HOOD through step 5:
Note: Lab coats and safety goggles should be worn while working with phenol/chloroform.
- Add 250 ul of Trizol reagent to the cell extract (Trizol is the Invitrogen brand name. The reagent contains phenol and guanidine isothiocyanate). Mix by inversion a few times and let incubate at room temperature for 5 minutes in the hood.
- Add 100 ul chloroform to each sample. Mix by shaking for 10 seconds and incubate for 2 minutes in the hood.
- Spin at top speed in a microfuge at 4°C for 15 minutes.
- Move the clear aqueous phase and white interphase to a new microfuge tube. DO NOT pipette any of the pink phenol (bottom layer). It is OK to leave a little of the aqueous phase behind to prevent getting any of the phenol (but not TOO much).
- Dispose of the phenol in the marked container in the hood. Put the empty microfuge tube in the marked bio-hazard bag.
- Estimate how much liquid is in your microfuge tube.
- Add 14 ul of 5M NaCl to your supernatant.
- Add 2 volumes of ICE COLD ethanol to your sample.
- Mix by shaking for a few seconds. Look at your sample - you should see small bubbles throughout your sample. That is the DNA precipitating.
- Put your samples at -80°C for 20 minutes. This helps to further precipitate your DNA.
- Spin your samples in a microfuge at the highest speed for 20 minutes at 4°C to pellet the DNA.
- Remove the supernatant and put that in a fresh microfuge tube to save just in case.
- Look on the sides of your tubes - can you see a pellet?
- Wash the pellet by gently adding 700 ul of 70% ethanol to the side of the tube opposite your pellet.
- Spin for 3 minutes at top speed at 4°C to be sure your pellet was not dislodged.
- Remove the ethanol and discard - being careful not to disrupt the pellet.
- Leave the tube open and upside-down on a paper towel on your bench for about 5 minutes to allow the ethanol to evaporate.
- Add 40 ul of TE to your pellet.
- Pipette up and down gently a few times to resuspend the pellet.
Agarose Gel Electrophoresis Protocol
Prepare a sample for electrophoresis by mixing 10 μL of your genomic DNA with 2 μL of loading dye on a 1.0% agarose gel with Ethidium Bromide stain. Run the gel at 100V for 45 minutes. The gel will be photographed using UV light and the photo posted to Sakai.
Template for loading the gel
Lane 1: NEB 1 kb ladder
Lane 2: Control 0 hours
Lane 3: Experimental 0 hours
Lane 4: Control 2 hours
Lane 5: Experimental 2 hours
Lane 6: Control 4 hours
Lane 7: Experimental 4 hours
Lane 8: Control 6 hours
Lane 9: Experimental 6 hours
Lane 10: Control 8 hours
Lane 11: Experimental 8 hours
Lane 12: NEB 100 bp ladder
What size is genomic DNA? What does it look like on the gel? What happens to the genomic DNA over time?
Lab 8: Cell Culture
Lab 10: Apoptosis - Protein 1
Lab 11: Apoptosis - Protein 2
Lab 12: Imaging Presentations